Viral hemorrhagic septicemia virus (VHSV) is known as a pathogen that infects freshwater salmonid fishes, including Oncorhynchus mykiss, to cause serious viral diseases. The VHSV is an about 11,000-bp negative-strand RNA virus belonging to the genus Novirhabdovirus of the family Rhabdoviridae, together with other fish viruses (infectious hematopoietic necrosisvirus (IHNV) and hirame rhabdovirus (HIRRV)), and comprises six genes, i.e., nucleocapsid (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV), and polymerase (L) in the order of 3′-N-P-M-G-NV-L-5′ (Schutze et al., 1999; van Regenmortel et al., 2000; Trdo et al., 2005).
Since VHSV was first isolated from Oncorhynchus mykiss in Denmark in 1963 (Jensen, 1965; Wolf, 1988), it has been isolated from Gadus morhua (Jensen et al., 1979), Scophthalmus maximus (Schlotfeldt et al., 1991), Clupea harengus (Dixon et al., 1997), Merlangius merlangus (Mortensen et al., 1999) and the like in the Atlantic Ocean and the North Sea. The VHSV is known as a pathogen that causes diseases in both freshwater and seawater fishes. The VHSV was isolated from seawater fish and anadromous salmon not only in the European region, but also from the Western shore of the North America region in the latter half of 1980s, indicating that the virus is widely distributed in the marine environment (Hopper, 1989; Meyers and Winton, 1995).
In the Eastern Asia region, the VHSV was first detected in wild Paralichthys olivaceus in Wakasa Bay, Japan (Takano et al., 2000), after which damage to cultured Paralichthys olivaceus by VHSV was also reported (Isshiki et al., 2001). In South Korea, rhabdovirus diseases in cultured Paralichthys olivaceus prevail at similar times. A virus that causes the rhabdovirus diseases was examined, and as a result, it was found that the virus had the same genotype as that of the VHSV isolated from the North America region and Japan (Kim et al., 2003). Since then, the VHSV has been isolated from various seawater fishes and freshwater fishes, and the VHSV was reported to be a pathogen that annually causes the death of cultured Paralichthys olivaceus (Kim et al., 2004; Kim et al., 2009; Cho et al., 2012).
At present, the VHSV is classified as a notifiable disease by the Aquatic Animal Health Code of the World Organization for Animal Health (OIE) (OIE, 2013). In South Korea, the VHSV is an infectious aquatic organism disease stated in Article 2 of the Aquatic Life Disease Control Act, and is included in diseases to be subjected to preventive measures, and when the outbreak of VHSV infection and disease is detected, restriction on movement and disinfection action are needed. In recent years, due to the outbreak of various aquatic organism diseases and the increasing consumer's demand for food safety, the importance of aquatic animal disease control, surveillance and monitoring has increased worldwide. In addition, not only national surveillance and control actions, but also analysis of the results of surveillance and monitoring of regional and zoned aquatic organism diseases, play a very important role not only in providing useful data required for epidemiological investigation of aquatic organism diseases and disease control, but also in demonstrating a region free of a specific disease in the corresponding country (FAO, 2004). Thus, continued studies on sequence mutations of Korean VHSV isolates, sequence comparison with VHSV isolates isolated from other countries, and continued monitoring of VHSV, became more important.
The nucleotide sequences of the N and G genes of VHSV isolates identified worldwide were phylogenetically compared. As a result, it was found that these genes contained four genotype (I-IV) and several subgroups of genotype I (Ia to Ie) and genotype IV (IVa and IVb) (Snow et al., 1999; Einer-Jensen et al., 2004; Lumsden et al., 2007). Genotype I includes VHSV isolates isolated from various freshwater and seawater fishes in Europe and also includes many VHSV isolates isolated from seawater fishes in Baltic Sea, Skagerrak, Kattegat and English Channel (Dixon et al., 1997; Thiery et al., 2002; Einer-Jensen et al., 2004; Snow et al., 2004). Genotype II includes VHSV isolates isolated from seawater fishes in Baltic Sea (Snow et al., 2004), and genotype III includes VHSV isolates isolated from the North Sea and North Atlantic of England and Ireland and VHSV isolates isolated from rainbow trout in Western Norway (Einer-Jensen et al., 2004; Snow et al., 2004; Lopez-Vazquez et al., 2006; Dale et al., 2009). Genotype IV includes VHSV isolates isolated not only from North America's Pacific coast and Atlantic coast, but also from North America's Great Lakes region and the Asian region (Nishizawa et al., 2002; Kim et al., 2003; Elsayed et al., 2006; Gagne et al. 2007; Lumsden et al. 2007). Thus, it appears that the results of analysis of the genetic distance of VHSV and the results of phylogenetic analysis of VHSV are more related to geographical positions than host fish species.
Various methods for analyzing bacterial genotypes have been developed, and Sanger sequencing, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) techniques and the like have been used. However, these methods have still problems in that the analysis time is lengthy and analysis procedures are complicated. Additionally, the development of genotype and genetic markers that indicate pathogenicity is required. In addition, in order to analyze the pathogenicity depending on the genotype to determine the correlation therebetween, the development of genotype and genetic markers for rapid and convenient analysis and a method capable of easily identifying genetic markers is required.
In a previous study, the present inventors performed phylogenetic comparison of VHSV isolates isolated from Asia (J. Fish Pathol., 26(3):149-161). The results of the study revealed that Korean VHSV isolates had C-to-A mutation at residue 755 in the nucleotide sequence of the VHSV G-protein gene. However, in the above-described study, all the full-length open reading frames of the G-proteins of the VHSV isolates were amplified, and then the amplification products were cloned, and the purified plasmid DNAs were sequenced. Thus, there was a disadvantage in that large amounts of time and material are required.
Generally, peptide nucleic acid (PNA) is a DNA analogue having nucleic acid nucleotides connected by peptide bonds instead of phosphate bonds, and was first synthesized by Nielsen et al. in 1991. PNA is not found in nature and is artificially synthesized by chemical methods. PNA forms a duplex by its hybridization to a natural nucleic acid having a nucleotide sequence complementary thereto. When their lengths are equal, a PNA/DNA duplex is more stable than a DNA/DNA duplex, and a PNA/RNA duplex is more stable than a DNA/RNA duplex. Furthermore, since PNA has a single base mismatch that makes the duplex unstable, the ability of PNA to detect SNP (single nucleotide polymorphism) is better than that of natural nucleic acid. PNA is chemically stable and is also biologically stable because it is not degraded by nuclease or protease. Furthermore, PNA is one of substances that recognize genes, like LNA (locked nucleic acid) or MNA (morpholino nucleic acid), and has a backbone consisting of polyamide. PNA has advantages in that it has very high affinity and selectivity and is thermally and chemically highly stable so that it can be easily stored and cannot be readily degraded.
Accordingly, the present inventors have made extensive efforts to identify a Korea-specific nucleotide sequence by analyzing the nucleotide sequences of the glycoprotein (G) of VHSV isolates isolated from cultured Paralichthys olivaceus in Korea and phylogenetically comparing Korean VHSV isolates with previously reported Japanese and Chinese VHSV isolates, thereby providing useful data required for the surveillance, genetic diversity analysis, molecular dynamic characterization, monitoring and control of zoned aquatic organism diseases. As a result, the present inventors have found that when a genetic marker for determining the region-specific genotype of VHSV and a peptide nucleic acid (PNA) having a high binding affinity for the genetic marker DNA are used so as to exhibit different melting temperatures depending on the region-specific genotype, the region-specific genotype of VHSV can be determined in a simple, rapid and accurate manner, thereby completing the present invention.